Was measured and presented as Mean 6 SD from three separate BMS 5 site experiments. doi:10.1371/journal.pone.0066464.gobserved that combined knockdown of p21 and PUMA leads to formation of acini with filled lumen and acquisition of enhanced migratory activity (Figure 5). These results further confirm the role of p21 in EMT, but most importantly, uncover a novel function for PUMA as a determinant of EMT in the morphogenesis of mammary epithelial cells. It is known that Slug is a suppressor of PUMA [34] and knockdown of Slug promotes apoptosis by upregulation of PUMA [35,36]. Here, we found that PUMA-KD increases the expression of Slug. Thus, the mutual regulation between PUMA-KD and Slug upregulation represents a novel feed-forward loop. We postulate that in response to downregulation of PUMA, Slug expression is induced, which in turn further inhibits expression of PUMA. As a result, the signaling cascade for EMT is amplified. In addition, we 11967625 found that the levels of EMT markers (Snail-1, Slug and Twist) increased by knockdown of both p21 and PUMA are much higher than that by p21-KD and PUMA-KD alone. Moreover, the EMT morphology is profound in the cells with p21 PUMA-KD. In light of these observations, we speculate that PUMA and p21 are two important determinants for EMT in the aberrant morphogenesis of mammary epithelialcells, and that PUMA might cooperate with p21 to prevent EMT in mammary epithelial cells via repressing expression of these transcription factors. DN isoform of p73 possesses a dominant negative activity towards TAp73 and possibly p53 [37,38]. Overexpression of DNp73 downregulates target genes of TAp73 and wild-type p53, such as the death receptors CD95 and TRAIL-R2 [39]. Conversely, deficiency of DNp73 leads to increased expression of p21 and PUMA [7,40,41]. Significantly, inactivation of DNp73 was found to increase apoptosis in mouse brain development [41,42]. Here, we found that in DNp73 PUMA-KD cells, knockdown of DNp73 mitigates the effect of PUMA-KD on cell polarity and EMT. This may be partly because p21 expression is increased by DNp73-KD. Similarly, in DNp73 p21-KD cells, DNp73-KD increases PUMA expression to compensatorily alleviate EMT induced by p21-KD. Since DNp73 has its own distinct activities [18,19], the counteracting effect of DNp73-KD on EMT may be due to the fact that DNp73 is required for increased expression of the EMT inducers (Snail-1, Slug, and Twist) (Figure 7A ).PUMA and p21 Regulate Morphogenesis and EMTFigure 6. Knockdown of DNp73 counters the effect of PUMA-KD or p21-KD on MCF10A cell morphogenesis. A-F, Generation of MCF10A cells in which both DNp73 and PUMA were stably knocked down (A-C, clones #2 and #3) or DNp73 and p21 were stably knocked down (DF, clones #2 and #3). The levels of DNp73 mRNA were measured by RT-PCR (A and D). The protein levels of TAp73a (B and E), DNp73a (B and E), PUMA (C and F), and p21 (C and F) were measured by Western blotting with antibodies against TAp73, DNp73, p21, and PUMA, respectively. MCF10A cells were untreated or treated with 0.2 mM doxorubicin for 24 h and total RNAs and cell extracts were collected for RT-PCR and Western blotting, respectively. G-H, Representative images of MCF10A cells with DNp73 PUMA -KD (G) or with DNp73 p21-KD (H) in 2-D culture (a, 2006) and 3-D culture (b, 406; c, 1006). I and L, Representative confocal images of 1485-00-3 web cross-sections through the middle of acini stained with To-Pro-3 and antibody against E-cadherin in MCF10A cells with DNp73 PU.Was measured and presented as Mean 6 SD from three separate experiments. doi:10.1371/journal.pone.0066464.gobserved that combined knockdown of p21 and PUMA leads to formation of acini with filled lumen and acquisition of enhanced migratory activity (Figure 5). These results further confirm the role of p21 in EMT, but most importantly, uncover a novel function for PUMA as a determinant of EMT in the morphogenesis of mammary epithelial cells. It is known that Slug is a suppressor of PUMA [34] and knockdown of Slug promotes apoptosis by upregulation of PUMA [35,36]. Here, we found that PUMA-KD increases the expression of Slug. Thus, the mutual regulation between PUMA-KD and Slug upregulation represents a novel feed-forward loop. We postulate that in response to downregulation of PUMA, Slug expression is induced, which in turn further inhibits expression of PUMA. As a result, the signaling cascade for EMT is amplified. In addition, we 11967625 found that the levels of EMT markers (Snail-1, Slug and Twist) increased by knockdown of both p21 and PUMA are much higher than that by p21-KD and PUMA-KD alone. Moreover, the EMT morphology is profound in the cells with p21 PUMA-KD. In light of these observations, we speculate that PUMA and p21 are two important determinants for EMT in the aberrant morphogenesis of mammary epithelialcells, and that PUMA might cooperate with p21 to prevent EMT in mammary epithelial cells via repressing expression of these transcription factors. DN isoform of p73 possesses a dominant negative activity towards TAp73 and possibly p53 [37,38]. Overexpression of DNp73 downregulates target genes of TAp73 and wild-type p53, such as the death receptors CD95 and TRAIL-R2 [39]. Conversely, deficiency of DNp73 leads to increased expression of p21 and PUMA [7,40,41]. Significantly, inactivation of DNp73 was found to increase apoptosis in mouse brain development [41,42]. Here, we found that in DNp73 PUMA-KD cells, knockdown of DNp73 mitigates the effect of PUMA-KD on cell polarity and EMT. This may be partly because p21 expression is increased by DNp73-KD. Similarly, in DNp73 p21-KD cells, DNp73-KD increases PUMA expression to compensatorily alleviate EMT induced by p21-KD. Since DNp73 has its own distinct activities [18,19], the counteracting effect of DNp73-KD on EMT may be due to the fact that DNp73 is required for increased expression of the EMT inducers (Snail-1, Slug, and Twist) (Figure 7A ).PUMA and p21 Regulate Morphogenesis and EMTFigure 6. Knockdown of DNp73 counters the effect of PUMA-KD or p21-KD on MCF10A cell morphogenesis. A-F, Generation of MCF10A cells in which both DNp73 and PUMA were stably knocked down (A-C, clones #2 and #3) or DNp73 and p21 were stably knocked down (DF, clones #2 and #3). The levels of DNp73 mRNA were measured by RT-PCR (A and D). The protein levels of TAp73a (B and E), DNp73a (B and E), PUMA (C and F), and p21 (C and F) were measured by Western blotting with antibodies against TAp73, DNp73, p21, and PUMA, respectively. MCF10A cells were untreated or treated with 0.2 mM doxorubicin for 24 h and total RNAs and cell extracts were collected for RT-PCR and Western blotting, respectively. G-H, Representative images of MCF10A cells with DNp73 PUMA -KD (G) or with DNp73 p21-KD (H) in 2-D culture (a, 2006) and 3-D culture (b, 406; c, 1006). I and L, Representative confocal images of cross-sections through the middle of acini stained with To-Pro-3 and antibody against E-cadherin in MCF10A cells with DNp73 PU.